A shuttle vector plasmid has been constructed which can be used to study gene stability, rearrangement, and recombination in mammalian cells. The plasmid contains sequences derived from a bacterial plasmid, from SV40 virus, and a marker gene, galactokinase, which can be scored in the appropriate bacterial host. This construct replicates in mammalian cells and bacteria. An experimental protocol was designed in which mammalian cells were infected with the plasmid, replication permitted and then the plasmid DNA extracted from the cells. After purification and elimination of residual infectious DNA, the plasmid was introduced into a bacterial host which permitted the detection of the presence or absence of a functional galactokinase gene. With this assay it was possible to assess quantitatively the stability of the plasmid in the mammalian cells. It was found that approximately 1% of the progeny plasmids had lost a functional marker gene. The defective plasmids contained point mutations, deletions, and insertions of cell DNA. The hypermutagenesis was found with different methods of DNA infection. Further studies showed that the mutagenesis was a property of the plasmid sequences, and was not shared by cellular genes. The plasmid mutagenesis occurs largely before plasmid replication begins, although there is some postreplication mutagenesis.